Viral vectors are the most popular gene delivery vehicles in gene therapy studies to treat various kinds of cancer, monogenic, vascular and infectious diseases such as cystic fibrosis, coronary heart disease and acquired immune deficiency syndrome (AIDS). Over the last fifteen years, the use of viral vectors in human gene therapy clinical trials have increased tremendously comprising 70% of the total clinical trials conducted worldwide. Murine-derived retroviral vectors (RV) are the most commonly used vectors (27%) followed by adenovirus (AdV) (26%), pox virus (7.2%), vaccinia virus (4.7%), herpes simplex virus (3%) and adeno-associated virus (AAV) (2.5%). For RV, the primary reason for being attractive gene delivery vehicles are their ability to integrate efficiently into the host genome maintaining a long-term gene expression while for AdV is their ease of production at high titers. The helper dependent adeno-associated virus (AAV) is one promising vector primarily because it is non-pathogenic and has a wide variety of host range. Likewise, a helper dependent adenovirus (HDAdV) that is fully deleted of its viral genes and requires a helper (as the term implies) virus for replication has also been commonly used mainly because of safety as it is less immunogenic than the first generation adenovirus. And for the purpose of covering the scope of this work, an emerging virus that have shown potential as gene delivery vector efficiently transducing mammalian cells is the insect cell derived baculovirus (BV) carrying mammalian cell promoters commonly termed as “BacMam”. BV has advantages over the other vectors since they are able to accommodate large foreign genes, are produced at high titers and non-pathogenic. The use of viral vectors in gene therapy continues to hold promise for the future despite major setbacks that only motivated for the development of safer vector constructs and new production systems to obtain higher virus titers. A great deal of effort has also been dedicated to the advancement of manufacturing processes whereby physical methods to quantify viral particles played a major role. These methods allowed results to be obtained in a matter of minutes facilitating process development in complementary to the traditional titration assays for infectious particles assays taking several days before a result is obtained. Several of these assays currently used for the quantification of total vector particles are ion-exchange high performance liquid chromatography (IE-HPLC) [Shabram P W, et al., Hum Gene Ther. 1997; 8: 453-465; Klyushnichenko V, et al. J Chromatogr B Biomed Sci Appl. 2001; 755: 27-36; Transfiguracion J, et al., J Chromatogr B Analyt Technol Biomed Life Sci. 2004; 813: 167-173; and Debelak D, et al., J Chromatogr B Biomed Sci Appl. 2000; 740: 195-202]; absorbance at 260 nm (Abs260nm) in the presence of SDS [Maizel J V Jr, et al., Virology. 1968; 36: 126-36; and Maizel J V Jr, et al., Virology. 1968; 36: 126-36]; viral genome labeling upon viral destruction [Murakami P, McCaman M T. Anal Biochem. 1999; 274: 283-8]: enzyme linked immunosorbent assay (ELISA) [Grimm D, et al. Gene Ther. 1999; 6: 1322-1330]; dot blot assay [Drittanti L, et al., Gene Ther. 2000; 7: 924-929] and polymerase chain reaction (PCR) [Sanburn N, Cornetta K. Gene Ther. 1999; 6: 1340-1345; Clark K R, et al., Hum Gene Ther. 1999; 10: 1031-1039; Carmo M, et al., J Virol Methods. 2004; 119: 115-119; Rohr U P, et al., J Virol Methods. 2002; 106: 81-88; and Veldwijk M R, et al., Mol Ther. 2002; 6: 272-278]. Although these methods provide rapid results, they also have their own disadvantages suffering either from non-specificity, insensitivity, laborious and expensive. For example, Abs260nm and genome labeling upon viral destruction are only applicable in the analysis of highly purified vector preparations devoid of contaminants. The presence of contaminants particularly host residual DNA could result in the overestimation of total particles since they also highly absorbs at Aba260nm. Quantification with crude preparations is not possible with these methods since there is no discrimination between the virus and the contaminants. IE-HPLC assays are specific because the virus is efficiently resolved from the rest of the sample components. However, these methods lack sensitivity requiring high virus titers for quantification. It has been reported that the quantification limits (QL) of these methods range from 2.5×107 and 3×108 viral particles per ml (VP/ml) for purified AdV5 and 1×108 VP/ml for virus lysates [Shabram P W, et al., Hum Gene Ther. 1997; 8: 453-465; and Transfiguracion J, et al., J Chromatogr B Biomed Sci Appl. 2001; 761: 187-194]. ELISA assays are time consuming and expensive, not suitable for the analysis of large volume of samples. PCR assays which are commonly used for the quantification of packaged vector genomes (vg's) are laborious and tend to suffer from non-specificity resulting in high variability of results requiring standardization on the method of operation for the comparison of inter-laboratory results [Veldwijk M R, et al., Mol Ther. 2002; 6: 272-278]. The validity of results obtained by this method also raised concerns due to the likeliness of unpackaged genome present in the preparation [Bartlett J S, et al., J Virol. 2000; 74: 2777-2785; and Ferrari F K, et al., J Virol. 1996; 70: 3227-3234].
Continuous efforts dedicated to the improvement of these methods and the development of new and better assays would only result in the better understanding of these vectors and better manufacturing processes.
Physical methods to quantify viral particles have become useful tools in the development of manufacturing processes of gene therapy vectors. Results can now be obtained in minutes facilitating process development. These methods though useful have their disadvantages suffering either from non-specificity, low sensitivity, laborious and expensive not suitable for routine use.
Accurate quantification of vector particles is critical in the design and execution of a successful pre-clinical and clinical gene therapy trials and experiments. Moreover, quantification assays for viral particles is an inherent and important part of a viral vector manufacturing process starting at the early stages of drug development. It is quite often overlooked at this stage but it is apparent that without a quantification assay, processes cannot be developed further. As previously discussed, particles quantification assays based on the physico-chemical characteristics of the virus have contributed to the speedy development of viral manufacturing processes. However, not one of these assays was actually able to fulfil an ideal routine assay that caters to all types of samples along the manufacturing process. For example, MV, RV and HDAdV are currently produced in low titers requiring the need for a more sensitive assay to be able to monitor a production. Crude virus preparations must be analysed with an assay that is able to discriminate the virus from the rest of the sample components or even better from the infectious to the non-infectious particles without compromising sensitivity and specificity. These are but a few examples of the limitations that some of the current methods are facing.
A method that would meet all these limitations would therefore be an ideal quantification assay.